1. Field of the Invention
The invention relates to polypeptides and peptides, particularly recombinant polypeptides and peptides, which can be used for the diagnosis of tuberculosis. The invention also relates to a process for preparing the above-said polypeptides and peptides, which are in a state of biological purity such that they can be used as part of the active principle in the preparation of vaccines against tuberculosis.
It also relates to nucleic acids coding for said polypeptides and peptides.
Furthermore, the invention relates to the in vitro diagnostic methods and kits using the above-said polypeptides and peptides and to the vaccines containing the above-said polypeptides and peptides as active principle against tuberculosis.
By "recombinant polypeptides or peptides" it is to be understood that it relates to any molecule having a polypeptidic chain liable to be produced by genetic engineering, through transcription and translation, of a corresponding DNA sequence under the control of appropriate regulation elements within an efficient cellular host. Consequently, the expression "recombinant polypeptides" such as is used herein does not exclude the possibility for the polypeptides to comprise other groups, such as glycosylated groups.
The term "recombinant" indeed involves the fact that the polypeptide has been produced by genetic engineering, particularly because it results from the expression in a cellular host of the corresponding nucleic acid sequences which have previously been introduced into the expression vector used in said host.
Nevertheless, it must be understood that this expression does not exclude the possibility for the polypeptide to be produced by a different process, for instance by classical chemical synthesis according to methods used in the protein synthesis or by proteolytic cleavage of larger molecules.
The expression "biologically pure" or "biological purity" means on the one hand a grade of purity such that the recombinant polypeptide can be used for the production of vaccinating compositions and on the other hand the absence of contaminants, more particularly of natural contaminants.
2. Description of the Prior Art
Tuberculosis remains a major disease in developing countries. The situation is dramatic in some countries, particularly where high incidence of tuberculosis among AIDS patients represents a new source of dissemination of the disease.
Tuberculosis is a chronic infectious disease in which cell-mediated immune mechanisms play an essential role both for protection against and control of the disease.
Despite BCG vaccination, and some effective drugs, tuberculosis remains a major global problem. Skin testing with tuberculin PPD (protein-purified derivative) largely used for screening of the disease is poorly specific, due to cross reactivity with other pathogenic or environmental saprophytic mycobacteria.
Moreover, tuberculin PPD when used in serological tests (ELISA) does not permit discrimination between patients who have been vaccinated by BCG, or those who have been primo-infected, from those who are developing evolutive tuberculosis and for whom an early and rapid diagnosis would be necessary.
A protein with a molecular weight of 32-kDa has already been purified from zinc deficient M. bovis BCG culture filtrate. This protein was identified as antigen 85A (De Bruyn J. et al., 1987, "Purification, partial characterization and identification of a 32-kDa protein antigen of Mycobacterium bovis BCG" Microb. Pathogen. 2:351). Its NH.sub.2 -terminal amino acid sequence (Phe-Ser-Arg-Pro-Gly-Leu) (SEQ ID NO:1) is identical to that reported for the .alpha.-antigen (antigen 85B) protein purified from M. bovis BCG (Wiker, H. G. et al., 1986, "MPB59, a widely cross-reacting protein of Mycobacterium bovis BCG" Int. Arch. Allergy Appl. Immunol. 81:307). The antigen 85-complex is present among different strains of mycobacteria (De Bruyn J. et al., 1989, "Effect of zinc deficiency of the appearance of two immunodominant protein antigens (32-kDa and 65-kDa) in culture filtrates of Mycobacteria" J. Gen Microbiol. 135:79). It is secreted by living bacilli as a predominant protein in normal Sauton culture filtrate and could be useful in the serodiagnosis of tuberculosis (Turneer M. et al., 1988, "Humoral immune response in human tuberculosis: immunoglobulins G, A and M directed against the purified P32 protein antigen of Mycobacterium bovis bacillus Calmette-Guerin" J. Clin. Microbiol. 26:1714) and leprosy (Rumschlag H. S. et al., 1988, "Serological response of patients with lepromatous and tuberculosis leprosy to 30-, 31- and 32-kilodalton antigens of Mycobacterium tuberculosis" J. Clin. Microbiol. 26:2200). Furthermore, the 32-kDa protein-induced specific lymphoproliferation and interferon-.gamma.(IFN-.gamma.) production in peripheral blood leucocytes from tuberculosis (Huygen K. et al., 1988, "Specific lymphoproliferation, .gamma.-interferon production and serum immunoglobulin G directed against a purified 32-kDa mycobacterial antigen (P32) in patients with active tuberculosis" Scand. J. Immunol. 27:187), and leprosy patients and from PPD- and lepromin-positive healthy subjects. Recent findings indicate that the amount of 32 kDa protein induced IFN-.gamma. in BCG-sensitized mouse spleen cells is under probable H-2 control (Huygen K. et al, 1989, "H-2-linked control of in vitro .gamma. interferon production in response to a 32-kilodalton antigen (P32) of Mycobacterium bovis bacillus Calmette-Guerin" Infect. Imm. 56:3196). Finally, the high affinity of mycobacteria for fibronectin is related to proteins of the antigen 85-complex (Abou-Zeid C. et al., 1988, "Characterization of fibronectin-binding antigens released by Mycobacterium tuberculosis and Mycobacterium bovis BCG" Infect. Imm. 56:3046).
Wiker et al. (Wiker H. G. et al., 1990, "Evidence for three separate genes encoding the proteins of the mycobacterial antigen 85 complex" Infect. Immun. 58:272) showed recently that the antigens 85A, B and C isolated from M. bovis BCG culture filtrate present a few amino acid replacements in their NH.sub.2 terminal region strongly suggesting the existence of multiple genes coding for these proteins. But, the data given for the antigen 85C of M. bovis BCG are insufficient to enable its unambiguous identification as well as the characterization of its structural and functional elements.
The gene encoding the 85A antigen from Mycobacterium tuberculosis has been described (Borremans L. et al., 1989, "Cloning, sequence determination and expression of a 32-kilodalton protein gene of Mycobacterium tuberculosis" Infect. Immun. 57:3123) which presented 77.5% homology at the DNA level within the coding region with the .alpha.-antigen gene (85B gene of M. bovis BCG, substrain Tokyo) (Matsuo K. et al., 1988, "Cloning and expression of the Mycobacterium bovis BCG gene for extracellular .alpha.-antigen" J. Bacteriol. 170:3847). Moreover, recently a corresponding 32-kDa protein genomic clone from a .lambda.gt11 BCG library (prepared from strain M. bovis BCG 1173P2) was isolated and sequenced. The complete sequence of this gene is identical with that from the 85A gene of Mycobacterium tuberculosis except for a single silent nucleotide change (De Wit L. et al., 1990, "Nucleotide sequence of the 32 kda-protein gene (antigen 85A) of Mycobacterium bovis BCG" Nucl. Ac. Res. 18:3995). Thus, it was likely, but not demonstrated, that the genome of M. bovis BCG contained at least two genes coding for antigen 85A and 85B respectively. As to the genome of the Mycobacterium tuberculosis and M. bovis, nothing was proved as to the existence of new genes, besides the genes coding respectively for 85A and 85B.